Alzheimer's disease (AD), the most common cause of dementia of the elderly, is a progressive neurodegenerative disorder characterized by the deposition of amyloid-beta (Abeta) and neurofibrillary tangles in the brain. Endoproteolytic cleavages of APP by beta-and gamma-secretases result in the generation of Abeta peptides. The exciting discoveries of beta-secretase and components of the gamma-secretase complex over the past several years provided opportunities to examine the physiological roles of BACE1 and Nicastrin (NCT) and to evaluate these proteins as therapeutic targets for AD. We created BACE1 null mice and demonstrated that BACE1 is the principal beta-secretase necessary to cleave APR to generate Abeta. In addition, we generated NCT null (NCT-/-) mice and NCT-/- cells and established that NCT is an integral member of the gamma-secretase complex. Taking advantage of our multidisciplinary group of talented investigators, we plan to extend our beta and gamma-secretase program to address several key issues outlined in the following Aims: 1) To determine whether deficits in synaptic functions or cognitive performance occur in BACE1-/-, BACE2-/-, or BACE1-/-;BACE2-/- mice;2) To develop a conditional tet-inducible BACE1 transgenic model to prospectively address the reversibility of Abeta induced abnormalities and the capacity of the brain to repair itself;3) To determine whether Abeta burden can be reduced in brains of mutant PS1;APP mice by genetically modulating the levels of BACE1 and/or components of the gamma-secretase complex;4) To test the hypothesis that Aph-1 and NCT are required to regulate the stability of each other to form a stable pre-complex for assembly of PS and Pen-2. In such a model, we suggest that the three mammalian Aph-1 homologues (Aph-1aL, Aph-1aS and Aph-1b) define a set of six distinct functional gamma-secretase complexes;5) To determine physiological role of NCT during post-natal development, maturation, and aging outside the central nervous system, NCT transgenic mice will be generated, characterized and crossbred to NCT-/- mice to complement the developmental defects in NCT-/- mice, and 6) To determine the roles of Aph-1a and Aph-1b by generation and characterization of mice and cells deficient in these components of the gamma-secretase complex. In concert, the Projects in this proposal are designed not only to examine the roles of BACE1, BACE2, and components of the gamma-secretase complex, but also allow a critical evaluation of these proteins as therapeutic targets in efforts to ameliorate Abeta amyloidosis in individuals with AD. PROJECT 1 P.I.: Donald L. Price, M.D. Title: BACE1 and BACE2 in Cognition and Models of Aa Amyloidosis Description (provided by applicant) With the discovery of BACE1 as the a-secretase involved in the generation of a-amyloid (Aa) peptides in Alzheimer's disease (AD), we embarked on a series of studies to examine the functional roles of this transmembrane aspartyl protease. We have provided evidence to support our hypothesis that the distributions and levels of BACE1 and BACE2, along with APR, are key determinants of selective vulnerability of brain to Aa amyloidosis. Importantly, deletion of BACE1 abolished Aa deposition and prevented cognitive deficits occurring in brains of mutant APP;PS1 mice. Although BACE1 null mice do not exhibit overt developmental abnormalities, our recent studies show that these animals do manifest alterations in performance on tests of cognition and emotion. The goal of Project 1 is to assess the functional roles of BACE1 and BACE2 and to evaluate critically BACE1 as a high priority therapeutic target for treatment of AD. Thus, studies in Aim 1 are designed to examine whether deficits in synaptic functions or cognitive/behavioral abnormalities occur in BACE1-l-, BACE2-l-, or BACE1-l- , BACE2-l- mice. In Aim 2, we plan to examine the link between abnormal accumulations of Aa peptides and synaptic abnormalities occurring in APPswe;PSl?E9 mice. These studies are critical for Aim 3, which are designed to assess the degree of reversibility/recovery following experimental reductions of BACE1 at different stages of Aa amyloidosis and degeneration. We anticipate that novel mechanism-based treatments such as BACE1 inhibitors will become available in the future, and it is therefore important to prospectively address the issues of the reversibility of Aa induced abnormalities and the capacity of the brain to repair itself. Investigations in Aim 3 are designed to determine to what extent Aa deposition and associated abnormalities can be reversed following reduction of BACE1 activity at various times after the initiation of Aa deposition. Taken together, results from these studies will provide important information regarding the physiological roles of BACE1 and BACE2 and allow a critical evaluation of BACE1 as a therapeutic target in efforts to reduce Aa burden in individuals with AD. Furthermore, these studies provide important information regarding potential mechanism based toxicities associated with anti-BACE1 therapy in humans that should be carefully monitored in clinical trials in the future.